pISSN 2233-8233 · eISSN 2233-8241 Clin Exp Reprod Med 2017;44(4):224-231
Impact of sperm DNA fragmentation on clinical in vitro fertilization outcomes
Hwa Young Choi1,2, Seul Ki Kim2,3, Seok Hyun Kim2,4, Young Min Choi2,4, Byung Chul Jee2,3
1Department of Obstetrics and Gynecology, Maria Fertility Hospital, Seoul; 2Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul; 3Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam; 4Department of Obstetrics and Gynecology, Seoul National University Hospital, Seoul, Korea
Objective: We studied the association between sperm DNA fragmentation (SDF) and several clinical in vitro fertilization outcomes.
Methods: We retrospectively analyzed 169 consecutive fresh IVF cycles. Semen was collected on the day of oocyte retrieval, and we assessed standard semen parameters and the SDF level (by terminal deoxynucleotidyl transferase dUTP nick-end labeling). Poor ovarian response (POR) was defined as the collection of three or fewer mature oocytes. Oocytes were inseminated by the conventional method or intracytoplasmic sperm injection.
Results: SDF did not affect the fertilization or pregnancy rate, but did have a significant effect on the miscarriage rate. In the miscarriage group (n=10), the SDF level was significantly higher (23.9% vs. 14.1%) and number of mature oocytes was significantly lower (4.3 vs. 7.6) than in the live birth group (n=45). Multiple regression analysis showed that SDF was an independent predictor of miscarriage (odds ratio, 1.051; 95%
confidence interval, 1.001–1.104). The cutoffs for the SDF level and number of mature oocytes that could predict miscarriage were >13% and
≤3, respectively. In the low-SDF group (≤13%), the miscarriage rate was similar in POR patients and those with a normal ovarian response (NOR; 14.2% vs. 4.3%). In the high-SDF group (>13%), the miscarriage rate was significantly higher in the POR group than in the NOR group (60.0% vs. 13.3%, p=0.045).
Conclusion: Our study demonstrated that a high SDF level (>13%) was associated with a high miscarriage rate, and that it mainly contributed to miscarriage in the POR group. The results suggest that SDF measurements should be considered in couples with POR in order to predict the prognosis of the pregnancy.
Keywords: Abortion; DNA fragmentation; Fertilization in vitro; Pregnancy; Spermatozoa
Introduction
Sperm DNA fragmentation (SDF) is not routinely assessed as part of semen analysis, but it can be offered as a special test [1]. Debate con- tinues on whether SDF measurements should become a part of the
routine fertility workup [2]. A number of studies have evaluated the influence of SDF levels on fertilization, embryo quality, and pregnan- cy outcomes, and five meta-analyses have been published [3-7].
Based on the high-quality evidence from the meta-analyses, high SDF levels appear to be associated with lower rates of clinical preg- nancy in standard in vitro fertilization (IVF) cycles, but not in intracy- toplasmic sperm injection (ICSI) cycles [3,5,6]. A significantly lower live birth rate in patients with a high SDF level has been reported, and this negative impact was prominent in IVF cycles [7]. Moreover, high SDF levels appear to be associated with higher miscarriage rates in ICSI cycles only [5] or in overall cycles [4], although some studies have reported no significant association with miscarriage rates [6].
Regarding the fertilization rate, one meta-analysis reported no asso- ciation with SDF levels in either standard IVF or ICSI cycles [3].
Received: Oct 4, 2017 ∙ Accepted: Oct 26, 2017 Corresponding author: Byung Chul Jee
Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 82 Gumi-ro 173beon-gil, Bundang-gu, Seongnam 13620, Korea Tel: +82-31-787-7254 Fax: +82-31-787-4054 E-mail: blasto@snubh.org
* This work was supported by the Korea Health Care Technology R&D Project, Ministry of Health and Welfare, Korea (No. A120043).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Although positive associations between SDF levels and several IVF outcomes have been reported, the majority of studies have not re- ported a clear cutoff SDF level predictive of poor fertilization, clinical pregnancy, live birth, or miscarriage rates.
When investigating the association of SDF levels with fertilization rates, previous studies have divided cycles into standard IVF groups and ICSI groups. However, since the indications for ICSI include not only male factors but also certain female factors, ICSI groups should be divided into male and female factor groups and evaluated separately.
In this study, we investigated the associations between SDF levels and several IVF outcomes, such as the fertilization rate, pregnancy rate, and miscarriage rate. For the associations between SDF levels and the fertilization rate, separate analyses were performed for stan- dard IVF patients and those who underwent ICSI due to male or fe- male factors, respectively.
Methods
1. Study population
The dataset for this retrospective study included 169 consecutive fresh IVF cycles performed between January 2012 and June 2014 at the Seoul National University Bundang Hospital. This study was ap- proved by the Institutional Review Board of the Seoul National Uni- versity Bundang Hospital (IRB No. B-1608-357-108). Written informed consents were obtained. The indications for IVF were unexplained in- fertility (n=78), tubal factor infertility (n=26), age factor infertility (n =20), endometriosis (n =18), uterine factor infertility (n =10), polycystic ovary syndrome (n=9), and male factor infertility (n=8).
The body mass index, basal serum level of follicle-stimulating hor- mone (FSH), and a random serum level of anti-Müllerian hormone in the female were recorded if they were measured within 2 months before starting the cycle.
2. Stimulation protocols and oocyte collection
Controlled ovarian stimulation was performed using recombinant FSH (Gonal-F; Serono, Geneva, Switzerland) with or without highly purified urinary gonadotropin (Menopur; Ferring, Malmo, Sweden) using a luteal long protocol with a gonadotropin-releasing hormone (GnRH) agonist (0.1 mg/day of Decapeptyl; Ferring) (n =10) or a GnRH antagonist protocol (0.25 mg/day of Cetrotide, Serono) (n=157). When two or more leading follicles reached a mean diame- ter of ≥18 mm, 250 μg of recombinant human chorionic gonadotro- pin (hCG; Ovidrel, Serono) was injected. Oocytes were retrieved 36 hours after the hCG injection. Poor ovarian response (POR) was de- fined as the collection of three or fewer mature oocytes. Metaphase I-derived in vitro matured oocytes were counted as mature oocytes, but germinal vesicle-derived oocytes were not.
3. Semen collection, measurement of SDF, and in vitro fertilization
Semen was collected on the day of oocyte retrieval and standard sperm quality was assessed in the raw semen (concentration, motili- ty, and normal form using strict criteria). Sperm quality was defined as normal (n =104) when the semen parameters were within the World Health Organization (WHO) reference values, regardless of the patient’s diagnosis. The subnormal group (n=43) included semen with parameters outside the WHO criteria, but not to the point of re- quiring ICSI, and the abnormal group (n=22) was made up of cases requiring ICSI.
The SDF level was measured by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) method in the raw se- men, following our previously reported institutional protocol [8]. Se- men samples were smeared on a silane-coated slide (DAKO Japan Co., Tokyo, Japan) and air-dried. Samples were fixed with 4% parafor- maldehyde for 1 hour at 15°C–25°C, washed with phosphate-buff- ered saline, and then were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (Sigma-Aldrich, St. Louis, MO, USA). A commer- cial apoptosis detection kit (in situ cell death detection kit; Roche Di- agnostics, Mannheim, Germany) was used to assess cell death in the samples according to the manufacturer’s instructions. Counterstain- ing was performed using a mounting medium with 4’,6-diamidino- 2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). The nuclei of sperm with fragmented DNA was stained green, whereas the nuclei of other cells were stained blue. Sperm heads with >50%
of the area stained green were considered positive for DNA fragmen- tation, following our previously reported institutional protocol [8]. At least 500 sperm cells were counted per experimental set, and the SDF level was determined as the percentage of sperm with frag- mented DNA.
The remaining semen was processed by a discontinuous gradient as described in the kit instructions (Sydney IVF density gradient me- dia; COOK, Brisbane, Australia). After initial centrifugation of the se- men (300×g for 5 minutes) to remove the seminal plasma, the ob- tained pellet was suspended in fresh Ham’s F10 medium (1.5 mL) supplemented with 10% synthetic serum substitute (SSS; Irvine Sci- entific, Santa Ana, CA, USA). Pre-washed semen (1.0 mL) was layered on the top of a discontinuous gradient in a 15-mL conical tube (40%/80%). The conical tube was centrifuged at 300×g for 5 min- utes, and the sperm cells collected from the bottom layer (80% layer) were washed twice by resuspension in 4 mL of Ham’s F10 medium and centrifugation (300×g for 5 minutes). After two rounds of cen- trifugation, the supernatant was removed and the pellet was resus- pended in 3 mL of Ham’s F10 medium supplemented with 10% SSS.
The resuspended pellet was later used in insemination.
The oocytes were inseminated by the conventional method
(n=69), by ICSI (n=95), or by split insemination (n=3), depending on the quality of the sperm and oocyte. ICSI was used in 22 cycles due to a male factor and in 73 cycles due to a female factor. Fertiliza- tion was confirmed by observing a two-pronuclear zygote on the day after oocyte retrieval. A low fertilization rate was defined as
≤75% in the current study, which corresponded to the 25th percen- tile of the overall fertilization rate.
4. Embryo transfer and confirmation of pregnancy
The embryos were transferred 3 or 5 days after oocyte retrieval.
Embryo quality was evaluated by morphological criteria based on the degree of fragmentation and the regularity of blastomeres on day 3 after fertilization. The embryos were graded as follows: grade A, 0% anucleate fragments, regularity of blastomeres, and no apparent morphological abnormality; grade B, <20% anucleate fragments, regularity of blastomeres, and no apparent morphological abnormal- ity; grade C, 20%–50% anucleate fragments, irregularity of blasto- meres, and no apparent morphological abnormality; and grade D,
>50% anucleate fragments, irregularity of blastomeres, and appar- ent morphological abnormalities. Blastocysts were evaluated on day 5 by the developmental stage and quality of the inner cell mass and trophectoderm. A good-quality blastocyst was defined as grade AA, AB, AC, BA, BB, or CA. Luteal phase support was performed using ei- ther a daily dose of 50 mg of progesterone in oil (Progest; Genefer, Seoul, Korea) or an 8% progesterone gel (Crinone, Serono), starting on the day of oocyte retrieval. Pregnancy was first assessed 14 days after oocyte retrieval by measuring the serum hCG level. In cases with positive hCG results, transvaginal ultrasonography was per- formed to confirm an intrauterine pregnancy and to identify the number of gestational sacs and the fetal heartbeat. Clinical pregnan- cy was defined as the presence of one or more gestational sacs. Mis- carriage was defined as pregnancy loss before 12 weeks of gestation.
5. Statistical analysis
All statistical analyses were performed using SPSS PASW ver. 18.0 (SPSS Inc., Chicago, IL, USA). When analyzing the association between
standard sperm quality and SDF levels, the data from 169 sperm sam- ples were used. The data from 164 cycles were used to analyze the fertilization rate, after excluding three cases with split insemination and two cases with no mature oocyte. Associations between SDF lev- els and pregnancy were analyzed in the 157 cycles in which embryo transfer was performed. Associations between SDF levels and miscar- riage were analyzed in the 55 cases that achieved clinical pregnancy.
Correlations were tested using the non-parametric Spearman rank test. The chi-square test was used to compare proportions between two groups. If the cell numbers were <5, the Fisher exact test was ap- plied to compare frequencies between groups. The medians of nu- meric data were compared using the Mann-Whitney U-test. Multiple regression analyses were performed for several numeric variables. A receiver operating characteristic (ROC) curve analysis was used to as- sess specific cutoff values for several numeric parameters. Results were considered significant with a two-tailed p-value <0.05.
Results
1. SDF levels and standard sperm parameters
The SDF levels ranged from 0.4% to 56.8% in the 169 sperm sam- ples (mean, 15.1%; median, 11.8%; standard deviation, 12.2%). They did not show a normal distribution. As shown in Table 1, in the over- all population, a positive correlation between SDF and the male part- ner’s age was found, but an inverse correlation was found between SDF and sperm motility. A multivariate analysis revealed that both the male partner’s age and motility were significantly associated with SDF (SDF=−0.444+[0.618×male partner’s age]−[0.162×mo- tility]). The positive correlation between SDF and the male partner’s age was prominent in those with normal sperm, while an inverse correlation between SDF and motility was prominent in the group with subnormal sperm. In the group with abnormal sperm, no signif- icant correlations were found between SDF and any of the standard sperm parameters.
In the group with normal sperm, the median SDF level (95% confi- dence interval [CI]) was 11.0% (8.5%–13%). The median SDF level
Table 1. Correlation coefficients for associations of the sperm DNA fragmentation level with standard sperm parameters
Variable Overall (n=169) Sperm quality
Normal (n=104) Subnormal (n=43) Abnormal (n=22)
Age of male partner (yr) 0.29a) 0.31a) 0.20 0.38
Volume (mL) –0.14 –0.09 0.04 –0.25
Concentration (million/mL) 0.06 0.01 0.33a) 0.27
Motility (%) –0.21a) –0.06 –0.53a) –0.31
Total motile sperm (million) –0.13 –0.08 –0.09 –0.14
Normal form (%) 0.07 0.01 0.22 –0.02
a)p<0.05 by the Spearman rank test.
was 13.0% (7.2%–18.7%) in the group with subnormal sperm and 14.9% (10.4%–29.8%) in the group with abnormal sperm. There was a significant difference between the median SDF level between the normal and abnormal sperm groups (p<0.05).
2. Impact of SDF on fertilization rate
SDF levels were not correlated with the fertilization rate in the over- all study population or in subgroups defined according to the insem- ination method or whether ICSI was performed due to a male or fe- male factor (Figure 1). In the three subgroups defined according to sperm quality, no significant correlations were found between SDF and the fertilization rate in the overall population, the standard IVF group, or the ICSI group with a female or male factor (Table 2).
A ROC analysis was performed to evaluate whether SDF levels could predict a low fertilization rate (i.e., ≤75%). The cutoff value for the SDF level was ≤5.1% in the overall population, ≤4.9% in the standard IVF group, >21.3% in the ICSI group, >21.3% in the female
factor ICSI group, and >12.9% in the male factor ICSI group. Howev- er, none of the values were statistically significant.
3. Impact of SDF on clinical pregnancy
The clinical pregnancy rate was 31% (40/129) in the day 3 transfer group and 53.5% (15/28) in the day 5 transfer group. SDF levels and other standard sperm parameters did not differ according to wheth- er pregnancy was achieved (data not shown). Two factors were sig- nificantly associated with pregnancy: female age (35 vs. 37 years, p=0.016) in the day 3 transfer group and the male partner’s age (34 vs. 38 years) in the day 5 transfer group (p=0.002).
4. Impact of SDF on miscarriage
Clinical pregnancy was achieved in 55 women, but 10 pregnancies ended in spontaneous miscarriage. In the miscarriage group, the SDF levels and serum FSH levels were significantly higher than in the live birth group, and the number of mature oocytes was significantly
100
50
0
Fertilization rate
ICSI cycle-female factor D
0 20 40 60 SDF
r=0.08 p=0.41 100
50
0
Fertilization rate
0 20 40 60 Overall
SDF
r=0.11 p=0.16 A
100
50
0
Fertilization rate
0 20 40 60 IVF cycle
SDF
r=0.09 p=0.41 B
100
50
0
Fertilization rate
0 20 40 60 ICSI cycle
SDF
r=0.07 p=0.44 C
Figure 1. Associations between sperm DNA fragmentation (SDF) levels and the fertilization rate in the overall study population and in sub- groups defined according to the insemination method or whether intracytoplasmic sperm injection (ICSI) was performed due to a female or male factor (r: correlation coefficients by the Spearman rank test). (A) Overall group, (B) con- ventional in vitro fertilization (IVF) group, (C) ICSI group, (D) ICSI due to female factor, (E) ICSI due to male factor.
100
50
0
Fertilization rate
0 20 40 60 ICSI cycle-male factor
SDF
r=0.04 p=0.99 E
Table 2. Association between the sperm DNA fragmentation level and the fertilization rate in the overall study population and sub-groups de-
fined according to sperm quality
Variable
Sperm quality
Normal Subnormal Abnormal
FR (%) No. r FR (%) No. r FR (%) No. r
Overall 100 102 –0.02 87.3 40 0.28 100 22 0.04
IVF group 84 50 0.05 75 19 0.27
ICSI group–female factor 100 52 –0.09 100 21 0.27
ICSI group–male factor 100 22 0.04
The median values for FR are given. Spearman rank test.
FR, fertilization rate; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection.
lower (Table 3).
A ROC analysis revealed that the cutoff values for predicting miscar- riage were an SDF level >13%, a basal serum FSH level >6.7 mIU/
mL, and ≤3 mature oocytes; all three cutoff values were statistically significant (Table 4). Neither the male partner’s age nor female age was a predictor of miscarriage. However, since both the male part- ner’s age and female age were possible confounders, multiple regres- sion analysis was performed, including five parameters (SDF level,
basal serum FSH level, number of mature oocytes, the male partner’s age, and female age). SDF was found to be the only significant predic- tive factor of miscarriage (odds ratio [OR], 1.051; 95% CI, 1.001–1.104).
5. IVF outcomes according to SDF and ovarian responsiveness Cycles with three or fewer mature oocytes usually indicate POR.
Since high SDF levels (>13%) and POR were significant predictive factors of miscarriage, we divided the whole population into a low- Table 3. Comparison between the live birth group and the miscarriage group
Variable Live birth (n=45) Miscarriage (n=10) p-value
Age of male partner (yr) 36.8 (35.4–38.3) 37.8 (34.6–41.1) NS
Volume (mL) 3.1 (2.7–3.5) 3.2 (2.5–3.9) NS
Concentration (million/mL) 133 (93–172) 135 (92–177) NS
Motility (%) 51 (45.9–56.0) 55.9 (41.8–70.0) NS
Total motile sperm count (million) 184 (128–240) 214 (144–284) NS
Normal form (%) 10.1 (8.2–11.9) 10.6 (6.2–15.0) NS
Sperm DNA fragmentation (%) 14.1 (10.3–17.9) 23.9 (12.9–35.0) 0.038
Age of female (yr) 33.9 (32.9–35.0) 36.1 (32.0–40.2) NS
No. of cycles 2 (1.6–2.5) 1.9 (0.7–3.1) NS
Basal serum FSH level (mIU/mL) 5.7 (4.9–6.5) 7.3 (5.9–8.8) 0.015
Random serum AMH level (ng/mL) 3.8 (2.7–4.8) 1.9 (0.6–3.2) NS
Dose of gonadotropin (ampule)a) 21.8 (20.0–23.7) 24.5 (20.1–28.9) NS
Peak serum estradiol level (pg/mL) 1,941 (1,450–2,431) 1,893 (732–3,053) NS
No. of mature oocytes 7.6 (6.0–9.2) 4.3 (1.6–7.0) 0.025
Method of insemination
IVF 28 5 NS
ICSI 16 5
Split insemination 1 0
Fertilization rate (%) 83 (77.3–88.7) 76.9 (58.9–95.0) NS
No. of day 3 transfers 32 8 NS
No. of day 5 transfers 13 2
No. of embryos transferred 2 (1.8–2.2) 1.6 (1.1–2.1) NS
Embryo quality (day 3 transfer only)
No. of grade A 1 (0.4–1.6) 1 (0.9–1.5) NS
No. of grades A+B 2 (1.7–2.2) 1 (0.9–1.8) NS
Endometrial thickness (mm) 8.8 (8.3–10.0) 10.6 (8.0–16.0) NS
Triple pattern endometrium (%) 95.5 90 NS
Values are presented as median (95% confidence interval). Mann-Whitney U-test.
NS, not significant; FSH, follicle-stimulating hormone; AMH, anti-Müllerian hormone; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection.
a)Each ampule contains 75 IU of gonadotropin.
Table 4. Predictive power of various factors for miscarriage
Variable Cutoff AUC 95% CI Sensitivity Specificity +LR –LR +PV –PV
Sperm DNA fragmentation (%) >13 0.713a) 0.575–0.827 80.0 62.2 2.12 0.32 32.0 93.3
Basal serum FSH level (mIU/mL) >6.7 0.768a) 0.614–0.883 77.8 79.4 3.78 0.28 50.0 93.1
No. of mature oocytes ≤3 0.733a) 0.597–0.843 70.0 77.8 3.15 0.39 41.2 92.1
Age of male partner (yr) >36 0.570 0.429–0.703 70.0 53.3 1.50 0.56 25.0 88.9
Age of female (yr) >35 0.622 0.481–0.749 60.0 73.3 2.25 0.55 33.3 89.2
AUC, area under the curve; CI, confidence interval; +LR, positive likelihood ratio; –LR, negative likelihood ratio; +PV, positive predictive value; –PV, negative pre-
dictive value; FSH, follicle-stimulating hormone.
a)p<0.05.
SDF (≤13%) and high-SDF (>13%) group and further divided them into POR and normal ovarian response (NOR) groups (Table 5). In the low-SDF group, the miscarriage rate was similar between the POR and NOR groups (14.2% vs. 4.3%). However, in the high-SDF group, the miscarriage rate was significantly higher in the POR group than in the NOR group (60.0% vs. 13.3%, p=0.045). The pregnancy rate did not differ across the four sub-groups.
Discussion
In the present study, SDF levels did not affect the fertilization rate or pregnancy rate in IVF/ICSI cycles, but did significantly affect the miscarriage rate. We found that the miscarriage rate was also affect- ed by POR, which is generally considered to be a poor prognostic factor. In this study, the pregnancy rate was significantly lower (25.4% vs. 42.2%, p=0.04) and the miscarriage rate was significantly higher (41.2% vs. 7.9%, p=0.01) in the POR group than in the NOR groups (Table 5). Nonetheless, multiple regression analysis revealed that SDF was the only significant predictive factor of miscarriage.
The miscarriage rate was highest in POR patients with a high SDF level (60%); this rate was significantly higher than that of NOR pa- tients with a low SDF level (4.3%) or NOR patients with a high SDF level (13.3%). This indicates that high SDF levels only contributed to miscarriage in the POR group. Therefore, SDF testing may be of par- ticular clinical significance for couples with POR.
When evaluating the effects of male parameters on IVF outcomes, it is important to control for female parameters, such as age, ovarian
reserve, and the number of retrieved oocytes. Dar et al. [9] evaluated the influence of high SDF levels on fertilization, clinical pregnancy, and miscarriage rates. Couples were matched by female age and se- rum anti-Müllerian hormone level since these variables could act as potential confounders. They showed that the fertilization and clinical pregnancy rates were similar between groups with a high SDF level (>50%) and groups with a low SDF level (≤15%). They showed a trend for a higher miscarriage rate in the high SDF group, but it did not reach statistical significance.
Jin et al. [10] evaluated the effect of SDF levels on IVF outcomes ac- cording to ovarian reserve. Reduced ovarian reserve was defined as basal FSH >10 mIU/mL, an antral follicle count <6, and a female age
≥38 years. They showed that SDF levels only had a significant im- pact on the clinical pregnancy and live-birth rates among women with reduced ovarian reserve; in women with normal ovarian re- serve, SDF levels had no impact.
In fact, disagreements exist about the association between SDF lev- els and miscarriage in IVF cycles. Two meta-analyses reported that high SDF levels were associated with higher miscarriage rates [4,5], but no association was reported in a recent meta-analysis [6]. The reasons for this disparity are largely unknown, but the use of differ- ent types of assays to assess DNA damage may be a reason. Robin- son et al. [4] found that the association between high SDF levels and miscarriage was strongest when using the TUNEL assay. The TUNEL assay directly quantifies DNA damage by the incorporation of la- beled dUTP into single- and double-stranded DNA breaks. It is gener- ally known to have a higher sensitivity and specificity for the detec- Table 5. Clinical outcomes according to the SDF and ovarian responsiveness
Variable SDF ≤13%
p-value SDF >13%
p-value
NOR (n=58) POR (n=36) NOR (n=35) POR (n=40)
Age of male partner (yr) 37 (36.2–38.5) 36 (35.8–39.0) NS 37 (36.6–39.5) 41 (39.5–42.6) 0.010 Age of female (yr) 34 (33.6–35.8) 34.5 (34.1–36.6) NS 35 (34.4–36.7) 39 (37.6–40.2) 0.001 Basal serum FSH level (mIU/mL) 4.9 (4.7–5.7) 6.6 (5.5–8.1) 0.012 5.9 (4.9–6.9) 8.1 (6.9–10.2) 0.007 Random serum AMH level (ng/mL) 3.6 (3.1–4.7) 0.8 (0.7–2.5) 0.001 2.6 (2.4–4.2) 1.1 (1.1–1.8) 0.001 Peak serum estradiol level (pg/mL) 1,998 (1,934–2,801) 698 (719–1,452) 0.001 1,942 (1,526–2,502) 651 (651–1,106) 0.001
No. of mature oocytes 8 (7.9–10.1) 2 (1.6–2.2) 0.001 6 (5.9–8.2) 2 (1.7–2.2) 0.001
No. of day 3 transfers 38 29 0.001 24 38 0.002
No. of day 5 transfers 19 0 9 0
No. of ET canceled cycles 1 7 0.009 2 2 NS
No. of embryos transferred 2 (2.0–2.3) 1 (1.3–1.9) 0.001 2 (1.9–2.3) 2 (1.6–2.1) NS
Embryo quality (day 3 transfer only)
No. of grade A 1 (1.1–1.6) 1 (0.4–1.0) 0.034 1 (0.7–1.3) 1 (0.4–0.9) NS
No. of grade A+B 2 (1.9–2.4) 1 (1.0–1.6) 0.002 2 (1.5–2.2) 1 (1.3–1.7) NS
Clinical pregnancy per transfer (%, n) 40.3 (23/57) 24.1 (7/29) NS 45.4 (15/33) 26.3 (10/38) NS Miscarriages per clinical pregnancy (%, n) 4.3 (1/23) 14.2 (1/7) NS 13.3 (2/15) 60.0 (6/10) 0.045 Values are presented as median (95% confidence interval). Mann-Whitney U-test.
SDF, sperm DNA fragmentation; NOR, normal ovarian response (>3 mature oocytes); POR, poor ovarian response (≤3 mature oocytes); FSH, follicle-stimulat- ing hormone; AMH, anti-Müllerian hormone; ET, embryo transfer.
tion of SDF than the sperm chromatin dispersion (SCD) test or sperm chromatin structure assay (SCSA). A comprehensive study reported that the cutoff value for defining male factor infertility was 20.1%
with a sensitivity of 0.764 and specificity of 0.952 when TUNEL analy- sis was applied [11].
Although an earlier meta-analysis reported no association between SDF levels and the fertilization rate, conflicting results have been re- ported on this topic [3]. High SDF levels have been reported to exert a negative effect on the fertilization rate, but studies have also found no association [9,12-17]. Some studies have reported a negative im- pact in standard IVF cycles but no impact in ICSI cycles [16,18]. In the current study, no associations between SDF levels and the fertiliza- tion rate were found in the whole population or in the five subgroups divided according to the insemination method, indication of ICSI, and semen quality.
It has been reported that DNA-damaged sperm cells could fertilize the oocyte regardless of the degree of DNA damage, but that embry- onic development and early pregnancy loss were closely related to the degree of damage [19]. Such a finding explains the absence of an association between SDF levels and the fertilization rate. However, high SDF levels have been reported to have an adverse impact on embryogenesis. Tesarik et al. [20] showed that high SDF levels were associated with a “late paternal effect” during the activation of male gene expression. Sperm cells with highly damaged DNA caused cer- tain paternal genome deficiencies and defective genomic activation within the embryo, thereby potentially exerting detrimental effects on late embryonic development [21,22].
The underlying mechanisms behind the close association between higher SDF levels and higher miscarriage rates in the POR group are largely unknown and require further investigation. Oocytes have been reported to possess the capability to repair damaged sperm DNA in murine models [19], and it was suggested that the effect of SDF levels on IVF/ICSI outcomes depended on oocyte quality [23].
The oocyte quality in the POR group might have been poor, meaning that oocytes in the POR group might have been less capable of re- pairing damaged sperm DNA than those in the NOR group.
The limitations of this study include the retrospective nature of the study design and small sample size. We used the TUNEL assay for SDF measurements, and therefore, a direct comparison with SDF as- sessed by SCD or SCSA was not possible.
In conclusion, no association was found between SDF levels and the fertilization rate or pregnancy rate in IVF/ICSI cycles. However, the SDF level significantly affected the miscarriage rate, especially in women with POR. These findings indicate that SDF testing should be performed in couples with POR to provide additional information on the prognosis of pregnancy.
Conflict of interest
No potential conflict of interest relevant to this article was reported.
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